Enhancing anti-tumor T cell responses using blocking antibodies against inhibitory T cell checkpoint receptors like PD-1 and CTLA-4, known as immune checkpoint blockade (ICB), is one of the most promising cancer treatment modalities. Despite very durable responses in various cancers, currently only a minority of patients benefits from ICB. Therefore, it is critical to understand why certain patients do not respond to ICB, and to identify ways to increase response rates. Recent evidence has shown that various metabolic pathways are crucial for T cell fate and function, and both checkpoint receptor signals and ICB affect T cell metabolism. Moreover, preliminary experiments show that metabolic pathways, specifically fatty acid oxidation (FAO) and oxidative phosphorylation, increase the cytotoxicity of CD8+ T cells that lack PD-1. In this proposal, the immunogenic MC38 murine colorectal carcinoma tumor model, which is sensitive to PD-1 pathway blockade, will be used to test the hypothesis that modulation of certain metabolic pathways can enhance anti-tumor T cell function and synergize with ICB. The proposed research comprises two arms. The first arm involves an extension of ongoing studies into the mechanisms that govern anti-tumor T cell function to the high-fat diet (HFD) context, a murine model of human obesity, to determine how this systemic metabolic perturbation affects anti-tumor T cell responses with and without ICB. This builds upon preliminary data suggesting that anti-tumor T cell function is altered in HFD. The second arm of this proposal involves a systematic investigation into which metabolic pathways modulate anti-tumor CD8+ T cell function, using a high-throughput in vitro system. This system will also be used to hone in on the mechanisms by which one metabolic pathway which was already shown to modulate CD8+ T cell cytotoxicity, FAO, affects cytotoxicity. In both arms of this proposal, metabolic pathways that regulate anti-tumor T cell function will first be identified, and then in vitro and in vivo manipulations of these pathways, using small molecules, metabolites and genetically engineered T cells, will reveal whether modulation of these pathways increases anti-tumor T cell function and synergizes with ICB. This research is novel, given that it is unknown whether obesity and HFD alter anti-tumor T cell responses, despite links between obesity and cancer, and between obesity and decreased immune function. Moreover, in contrast to the role of metabolic pathways in T cell activation and the development of effector cells, the importance of metabolic pathways for T cell effector function, especially cytotoxicity, has not been systematically assessed. This goals of this research, investigating how metabolic pathways regulate anti-tumor T cell function and responsiveness to ICB, are to identify metabolic pathways that promote anti-tumor T cell responses and to define metabolic interventions that synergize with ICB.